"Behe ...
Brings up the clotting cascade, and points out an error made by Russ Doolittle
in a Boston Review article in 1997. Doolittle mischaracterized a 1996 paper on
double knockout mice lacking two proteins in the clotting cascade. Behe
introduces a theme for the evening: if Russ Doolittle, the world's expert,
makes mistakes about the clotting cascade, then there is no evidence that it
arose by DEM."

So,
again, Behe has brought up Doolittles alleged "mistake" several times. Would it
surprise you that Behe is being less than forthright?

"Let me
conclude by mentioning that support for the Yin and Yang scenario is now coming
from another quarter. Thus, it has become possible during the last decade to
"knock out" genes in experimental organisms. "Knockout mice" are now a common
(but expensive) tool in the armamentarium of those scientists anxious to cure
the world's ills. Recently the gene for plaminogen was knocked out of mice,
and, predictably, those mice had thrombotic complications because fibrin clots
could not be cleared away. Not long after that, the same workers knocked out
the gene for fibrinogen in another line of mice. Again, predictably, these mice
were ailing, although in this case hemorrhage was the problem. And what do you
think happened when these two lines of mice were crossed? For all practical
purposes, the mice lacking both genes were normal [6]. Contrary to claims about
irreducible complexity, the entire ensemble of proteins is not needed. Music
and harmony can arise from a smaller orchestra. No one doubts that mice deprived of these two genes would be compromised
in the wild, but the mere fact that they appear normal in the laboratory setting is a striking example of
the point and counterpoint, step-by-step scenario in reverse!"(emphasis added
by IFM)

Now,
let's be clear what Doolittle is stating here.
1) The example shows the interaction between regulatory proteins in the
cascade. The modern clotting system is a balance between pro-coagulation
factors and anti-coagulation factors (the Yin and Yang as he calls it)

The main
point was to demonstrate that the delicate balance of forward and reverse
reactions that regulate blood clotting came about in a step-by-step fashion. I
summarized events with the metaphor of Yin and Yang, and emphasized that other
similar point-and-counterpoint comparisons could be made.

2) The
example shows that simpler systems than the full system can work in laboratory
situations.
3) The mice in the example wouldn't survive in the wild

Note
that Doolittle is clearly
relating the recovery in the double knockout mice to the excessive coagulation
defects in the plasminogen knockout mice, not the overall life history of the
mice (see below for some of the amazing results from the actual paper).

Behe
has responded to Doolittle in several fora, saying he made a mistake. In this
article Behe, quotes part of the paragraph. Behe carries on as if
Doolittle thought these mice were exactly the same as wild-type mice in all
respects. However, Behe omits the last
section of Doolittle's paragraph, with its qualifications and statement
that these mice are by no means identical to ordinary mice and wouldn't survive
in the wild. At the most Doolittle is guilty of using jargon (compromised
rather than die; however, researchers still tend to write that animals
were sacrificed, rather than animlas were killed, when describing experiments).

It is
Behe who is doing the mischaracterizing, not Doolittle.

Now,
I've read Bugge et al, and I
think Behe's statement not only mischaracterizes Doolittle, but also
mischaracterizes the Bugge et al.
paper.

Clotting
occurs by a cascade of reactions initiated by either tissue damage (the
extrinsic pathway) or contact with surfaces such as the basement membranes of
blood vessels (the intrinsic or contact pathway). The ultimate effect of this
cascade is to convert the soluble protein fibrinogen to the insoluble protein fibrin,
which forms the basis of the clot that stops bleeding.

Note
that this is the critical final step in clotting. Now, what happens when we
knockout the gene for fibrinogen, making the mice unable to form fibrin (and
hence clots)? Nothing much really. In the laboratory the fibrinogen knockout
mice (Fib-/-) live normal lives with minor defects. They can't carry a
pregnancy beyond mid-term, but they should
be dead from massive bleeding a day or two after birth, not living well into
breeding age. The reason is that the clotting system not only stops bleeding
from cuts and wounds, it also maintains the integrity of the blood vessels.
Without a low level of clotting activity, the blood vessels (mostly the small
ones) leak [note1].

Not
only do the Fib (-/-) mice fail to die catastrophically after birth, but they
fail to bleed to death from full skin thickness incisions. Remember, that
fibrin is the ultimate product of the clotting pathway, yet these mice are
coping well in the laboratory (in the wild they would probably be dead rapidly,
researchers with scalpels are kinder than cats, pointy sticks and other natural
hazards). Why is this so? Almost certainly because platelets are plugging the
blood vessels and slowing the bleeding until ordinary serum proteins glob up
[2]. This is of interest because this is basically the mechanism that primitive
chordates such as tunicates use.

These
animals have a low-pressure blood circulation system and use circulating
haemocytes (a bit like white blood cells) to plug up any wounds. I'd like to
emphasize that the vertebrate clotting system did not evolve in modern mammals
with high-pressure circulatory systems, but developed between the tunicates
(which have no readily identifiable components of the modern coagulation system)
and cartilaginous-jawed fish (which have most of the pathways)[3]. It can be
readily seen that a generalized "globbing system", well short of the modern
coagulation system would be an advantage over the tunicate hemolymph system,
with out requiring the specificity of the modern vertebrate coagulation system
[note2].

When
the blood vessel is repaired the fibrin has to be cleared away, this is
performed largely by plasmin, generated from its inactive precursor,
plasminogen. This is part of the balanced "Yin/Yang" system that
Doolittle talks about. Plasminogen knockout mice (Pl-/-) have no plasminogen.
The Pl(-/-) mice are very sick.
They fail to gain weight, have very poor wound healing, have numerous
pathological changes and all die within 300 days [1].

So
what happens when we knock out both plasminogen and fibrinogen? With both of
these essential components gone the mice should be even sicker, right? Wrong!
Plasminogen/Fibrinogen double knockouts gain weight almost at the rate normal
mice do, have wound healing almost indistinguishable from normal mice and live
for virtually the same time as normal animals. They do have a few pathological
changes, but as Doolittle says, "for all practical purposes" these animal are
normal (see the following figures, they are pretty amazing).

Wound
healing, the plasminogen knockout mice (triangles) heal very slowly, while the
Pl(-/-),Fib(-/-) mice (diamonds) not only fail to bleed to death immediately,
but heal almost as fast as normal mice (squares).

Now,
let me emphasize again that these animals fail to bleed to death
catastrophically or do any of the horrible things that one would expect from
Behe's "irreducibly complex" scenario. As Doolittle says, this shows the
Yin/Yan aspect of coagulation, with checks and balances that can be built up
step by step.

Now,
fibrinogen knockout mice cannot carry a pregnancy beyond day 10, neither can
the double knockout mice. This is not particularly significant when by Behe's
ideas these mice should be dead shortly after birth, but conspicuously
aren't. In the wild, they wouldn't be able to breed, but in the wild they
would be dead long before breeding age. The way Doolittle frames his
brief discussion, given the limitations of a newspaper article ("No one doubts
that mice deprived of these two genes would be compromised in the wild") is
perfectly compatible with the facts of the Bugge et al., paper.

Doolittle
never claimed they were ("No one doubts that mice deprived of these two genes
would be compromised in the wild"), after all clotting evolved in
protovertberates with low-pressure blood systems, not modern vertebrates [3,4].
What Doolittle was pointing to was how the systems interact with each other,
and how systems could be built up piecemeal. Behe seizes on a minor point, only
made possible by not quoting the full text of Doolittle's paragraph, and
ignores the fact that fibrinogen deficient mice don't bleed to death
immediately after birth, as his scheme would predict.

In
summary, Behe misrepresents Doolittle. Basic courtesy would at least
require Behe to include the full
Doolittle paragraph in his article, let alone ceasing to claim Doolittle "made
a mistake." [note 3].

Notes

[note1]
Thrombin knockout mice are embryonic lethal. However, it turns out that
thrombin leads a double life as an embryonic tissue remodelling enzyme. It is
entirely possible that thromin was co-opted from development remodelling
enzymes[2]. There is a lot of evidence that he complement system originated by
co-opting tissue remodelling enzymes.

[note2]
While its is easy to imagine a trypsin like enzyme chopping bits off previously
soluble protein to make it insoluble, making a protoclot, one issue that has
been raised is how do you stop this primitive trypsin-like enzyme from clotting
the entire blood, rather than where it is needed. A clue comes from the
primitive chordate Amphioxis. It has trypsin circulating in its haemolymph (it
doesn't have blood like vertebrates), when the animal is injured, trypsin
cleaves a bit off an enzyme activating it, which in turn produces a polymeric
melanin which acts as a kind of glue to immobilize bacteria. Why isn't trypsin
activating this enzyme all the time? Trypsin is only activated in the presence
of calcium released by tissue damage. This is a primitive, one step version of
the calcium-activated clotting cascade.

[note 3] At least one other webpage has Behe partly quoting
Doolittle's statement and omitting the qualifier, as well as proceeding as if
the qualifier wasn't there. Behe also repeats it often in lectures and talks.

"Soon after the book was published, its thesis was
challenged by the leading expert in America on cell structure, Dr. Russell
Doolittle at the University of California. He cited a scientific study
supposedly disproving irreducible complexity. Behe immediately researched it
and found it proved just the opposite: It confirmed him. So Behe went back to
Dolittle. In a phone conversation, Doolittle admitted he was wrong, but he has
never made a public retraction."

Is this story true? Far from it. Mark Perakh emailed Dr.
Doolittle, who generously allowed his reply to be quoted.

Doolittle wrote:

"Thanks for sending
me the latest on the desperate tactics that these people avail themselves of.
Others have written me in the past about my alleged "concession." What happened
is that Behe emailed me after my article appeared and challenged my tail-end
assertion about the Bugge et al paper. I went back and read it more carefully,
and indeed, I had overstated one aspect. It was inadvertant, and I apologized.
Ah, rue the day with minds like theirs!

In my paper I raised the point of how mice without
plasminogen were severely ill. Mice without fibrinogen are also unwell,
although not as badly off as the plasminogen-lackers. However, the big point
was that mice lacking both genes were considerably better off than the ones
that only lacked plasminogen. I had said these mice were "..for all practical
purposes..normal," although I went on to qualify the statement about how the
mice would doubtless be compromised in the wild. In fact, the paper makes it
clear that the mice are not "normal" in that they don't breed well, even though
the individuals are not unwell. My original point, lost in all the persiflage,
was that there is a "point and counterpoint" in the evolution of these two
genes, as there is in the evolution of many interacting systems.

It's
truly annoying that the creationists ("Intelligent Designers") get so much
mileage out these side-issues and refuse to confront the main message.
Regards,
Russell Doolittle"

[IFM is not an active researcher in the coagulation field,
but my first ever research project was studying the effects of snake venom on
coagulation, I still have the platelet electron micrographs taken nearly 20
years ago.]